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The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors.

Vanden Bergh P, Heller M, Braga-Lagache S, Frey J - Proteome Sci (2013)

Bottom Line: Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria.T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619.Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Veterinary Bacteriology, University of Bern, Länggassstrasse 122, Bern, Switzerland. joachim.frey@vetsuisse.unibe.ch.

ABSTRACT

Background: Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth.

Results: Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected.

Conclusions: We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

No MeSH data available.


Related in: MedlinePlus

Protein abundance of A. salmonicida appendages. The diagram represents total PMSS values of A. salmonicida secretion systems (T1- to T6SS), flagella (lateral and polar), pili (T1 pilus [Fim], T4 pili [Tap, Flp and Msh] and fimbriae (MatB). Only the structural components were taken into account for the T3SS. Logically, the strongest difference in protein amount was observed for the T3SS. No differences were observed in pellets for T1- (103 ABC-transporters) and T2SS. In SNs, the strong difference between wt and mutant strains for T1SS values is due to the higher amount of VapA in mutant SNs.
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Figure 4: Protein abundance of A. salmonicida appendages. The diagram represents total PMSS values of A. salmonicida secretion systems (T1- to T6SS), flagella (lateral and polar), pili (T1 pilus [Fim], T4 pili [Tap, Flp and Msh] and fimbriae (MatB). Only the structural components were taken into account for the T3SS. Logically, the strongest difference in protein amount was observed for the T3SS. No differences were observed in pellets for T1- (103 ABC-transporters) and T2SS. In SNs, the strong difference between wt and mutant strains for T1SS values is due to the higher amount of VapA in mutant SNs.

Mentions: The mutant strain might continue to release these T3SS components in SNs, either from the resting structural T3SS components or by an alternative secretion pathway. Recent publications argue that the T3SS arose from an exaptation of the flagellum, i.e. the recruitment of part of the flagellum structure for the evolution of the new protein delivery function [33,34] and, the secretion of T3SS effectors through flagella in the extracellular medium has been described in other bacteria [35,36]. The secretion of effectors/translocators by this process is unlikely in A. salmonicida given that functional lateral and polar flagella were not detected (Figure 4), thus confirming the results of studies showing that operons coding for A. salmonicida flagella contain several mutations [12]. However, we could imagine that FlhA (ASA_1351, polar flagella) and/or LfhA (ASA_0352, lateral flagella), showing respectively 56% and 55% of similarity with AscV might partially supply the function of this T3SS component. Such possible interactions between FlhA and the T3SS have been described in Chlamydia pneumonia[37]. While no mutations are predicted in these genes in A. salmonicida their expression was not detected in our pellets, but we cannot exclude that they were expressed below the detection limit of our system as our proteomic analysis did not cover the total proteome (59% of proteins common to all Aeromonas sp. were identified). Another possibility is that two mechanisms of effectors/translocators secretion operate in parallel along phases of growth, the first would be actively dependent on intact T3SSs while the second, clearly less efficient, would explain the progressive accumulation of effectors/translocators in the extracellular medium of the mutant strain. The alternative secretion of T3SS effectors through classical/unclassical pathways has never been described contrary to major constituents of the OM ring which are transported to the periplasm by the Sec-dependent secretion pathway [38]. Furthermore, the presence of T3SS effectors in the periplasma [39] and OMVs [40] has rarely been described. Another possibility might be the formation of double-bilayer OMVs (diameter from 100 to 250 nm) containing cytosolic components, as recently described in Shewanella[41], but GroEL would have been detected in SNs. Another study showed that in the absence of the host cell, at least YopH (homologous to AopH), YopE (AexT) and YopB/YopD translocators were excreted homogeneously at the Yersinia surface without physical association with the injectisome [42]. While the T3-dependent secretion of effectors in SNs is well-characterized, it is not known if these OM-associated effectors are excreted at the bacterial surface through a T3SS-dependent pathway. As a result, further investigations are necessary to clarify this point.


The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors.

Vanden Bergh P, Heller M, Braga-Lagache S, Frey J - Proteome Sci (2013)

Protein abundance of A. salmonicida appendages. The diagram represents total PMSS values of A. salmonicida secretion systems (T1- to T6SS), flagella (lateral and polar), pili (T1 pilus [Fim], T4 pili [Tap, Flp and Msh] and fimbriae (MatB). Only the structural components were taken into account for the T3SS. Logically, the strongest difference in protein amount was observed for the T3SS. No differences were observed in pellets for T1- (103 ABC-transporters) and T2SS. In SNs, the strong difference between wt and mutant strains for T1SS values is due to the higher amount of VapA in mutant SNs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852671&req=5

Figure 4: Protein abundance of A. salmonicida appendages. The diagram represents total PMSS values of A. salmonicida secretion systems (T1- to T6SS), flagella (lateral and polar), pili (T1 pilus [Fim], T4 pili [Tap, Flp and Msh] and fimbriae (MatB). Only the structural components were taken into account for the T3SS. Logically, the strongest difference in protein amount was observed for the T3SS. No differences were observed in pellets for T1- (103 ABC-transporters) and T2SS. In SNs, the strong difference between wt and mutant strains for T1SS values is due to the higher amount of VapA in mutant SNs.
Mentions: The mutant strain might continue to release these T3SS components in SNs, either from the resting structural T3SS components or by an alternative secretion pathway. Recent publications argue that the T3SS arose from an exaptation of the flagellum, i.e. the recruitment of part of the flagellum structure for the evolution of the new protein delivery function [33,34] and, the secretion of T3SS effectors through flagella in the extracellular medium has been described in other bacteria [35,36]. The secretion of effectors/translocators by this process is unlikely in A. salmonicida given that functional lateral and polar flagella were not detected (Figure 4), thus confirming the results of studies showing that operons coding for A. salmonicida flagella contain several mutations [12]. However, we could imagine that FlhA (ASA_1351, polar flagella) and/or LfhA (ASA_0352, lateral flagella), showing respectively 56% and 55% of similarity with AscV might partially supply the function of this T3SS component. Such possible interactions between FlhA and the T3SS have been described in Chlamydia pneumonia[37]. While no mutations are predicted in these genes in A. salmonicida their expression was not detected in our pellets, but we cannot exclude that they were expressed below the detection limit of our system as our proteomic analysis did not cover the total proteome (59% of proteins common to all Aeromonas sp. were identified). Another possibility is that two mechanisms of effectors/translocators secretion operate in parallel along phases of growth, the first would be actively dependent on intact T3SSs while the second, clearly less efficient, would explain the progressive accumulation of effectors/translocators in the extracellular medium of the mutant strain. The alternative secretion of T3SS effectors through classical/unclassical pathways has never been described contrary to major constituents of the OM ring which are transported to the periplasm by the Sec-dependent secretion pathway [38]. Furthermore, the presence of T3SS effectors in the periplasma [39] and OMVs [40] has rarely been described. Another possibility might be the formation of double-bilayer OMVs (diameter from 100 to 250 nm) containing cytosolic components, as recently described in Shewanella[41], but GroEL would have been detected in SNs. Another study showed that in the absence of the host cell, at least YopH (homologous to AopH), YopE (AexT) and YopB/YopD translocators were excreted homogeneously at the Yersinia surface without physical association with the injectisome [42]. While the T3-dependent secretion of effectors in SNs is well-characterized, it is not known if these OM-associated effectors are excreted at the bacterial surface through a T3SS-dependent pathway. As a result, further investigations are necessary to clarify this point.

Bottom Line: Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria.T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619.Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Veterinary Bacteriology, University of Bern, Länggassstrasse 122, Bern, Switzerland. joachim.frey@vetsuisse.unibe.ch.

ABSTRACT

Background: Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth.

Results: Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected.

Conclusions: We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

No MeSH data available.


Related in: MedlinePlus